Methods and compositions for delivery of agents across the blood-brain barrier

ABSTRACT

Sequences that enhance permeation of agents into cells and/or across the blood brain barrier, compositions comprising the sequences, and methods of use thereof.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.17/258,846, filed on Jan. 8, 2021, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2019/041386, filed on Jul. 11, 2019, which claims the benefit ofU.S. Provisional Application Ser. No. 62/696,422, filed on Jul. 11,2018. The entire contents of the foregoing are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 2, 2021, isnamed Sequence_Listing.txt and is 58,834 bytes in size.

TECHNICAL FIELD

Described herein are sequences that enhance permeation of agents acrossthe blood brain barrier, compositions comprising the sequences, andmethods of use thereof.

BACKGROUND

Delivery of therapeutic agents, including gene therapy reagents, is animpediment to development of treatments for a number of conditions. Theblood-brain barrier (BBB) is a key obstacle for drug delivery to themammalian central nervous system (CNS), particularly for delivery to thehuman brain, to treat conditions including neurodegenerative diseasessuch as Parkinson's disease; Alzheimer's disease; Huntington's disease;Amyotrophic lateral sclerosis; and Multiple sclerosis.

SUMMARY

The present invention is based on the development of artificialtargeting sequences that enhance permeation of agents into cells andacross the blood brain barrier.

Thus provided herein is an AAV capsid protein, e.g., an engineered AAVcapsid protein, comprising a targeting sequence that comprises at leastfour contiguous amino acids from the sequence TVSALFK (SEQ ID NO:8);TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).In some embodiments, the AAV capsid protein comprises a targetingsequence that comprises at least five contiguous amino acids from thesequence TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ IDNO:83); or KFLASVT (SEQ ID NO:84). In some embodiments, the AAV capsidprotein comprises a targeting sequence that comprises at least sixcontiguous amino acids from the sequence TVSALK (SEQ ID NO:4); TVSALFK(SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

In some embodiments, the AAV is AAV9; other AAV as known in the art(e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof and others asknown in the art or described herein) can also be used.

In some embodiments, the AAV capsid protein comprises AAV9 VP1 (e.g.,SEQ ID NO:85).

In some embodiments, the targeting sequence is inserted in the capsidprotein at a position corresponding to between amino acids 588 and 589of SEQ ID NO:85.

Also provided herein are nucleic acids encoding the AAV capsid proteinscomprising a targeting sequence as described herein.

In addition, provided herein is an AAV comprising a capsid proteincomprising a targeting sequence as described herein. In someembodiments, AAV further comprises a transgene, preferably a therapeuticor diagnostic transgene. Therapeutic transgenes can include, e.g., cDNAsthat restore protein function, guide RNA for gene editing, RNA, ormiRNA.

Also provided herein are targeting sequences comprising V[S/p][A/m/t/]L(SEQ ID NO:79), TV[S/p][A/m/t/]L (SEQ ID NO:80), TV[S/p][A/m/t/]LK (SEQID NO:81), or TV[S/p][A/m/t/]LFK. (SEQ ID NO:82). In some embodiments,the targeting sequence comprises VPALR (SEQ ID NO: 1); VSALK (SEQ IDNO:2); TVPALR (SEQ ID NO:3); TVSALK (SEQ ID NO:4); TVPMLK (SEQ ID NO:12); TVPTLK (SEQ ID NO:13); FTVSALK (SEQ ID NO:5); LTVSALK (SEQ IDNO:6); TVSALFK (SEQ ID NO:8); TVPALFR (SEQ ID NO:9); TVPMLFK (SEQ IDNO:10) or TVPTLFK (SEQ ID NO:11). Also provided are fusion proteinscomprising the targeting sequences linked to a heterologous (e.g.,non-AAV VP1) sequence, and AAV capsid proteins (e.g., AAV9 VPT)comprising the targeting sequence. In some embodiments, the targetingsequence is inserted in a position corresponding to amino acids 588 and589 of SEQ ID NO:85.

Additionally provided herein are nucleic acids encoding the targetingsequences, fusion proteins or AAV capsid proteins described herein, aswell as AAV comprising the capsid proteins comprising a targetingsequence. In some embodiments, the AAV further comprises a transgene,preferably a therapeutic or diagnostic transgene. Therapeutic transgenescan include, e.g., cDNAs that restore protein function, guide RNA forgene editing, RNA, or miRNA.

Further, provided herein are methods of delivering a transgene to acell, the method comprising contacting the cell with an AAV or fusionprotein described herein. In some embodiments, the cell is in a livingsubject, e.g., a mammalian subject. In some embodiments, the cell is ina tissue selected from the brain, spinal cord, dorsal root ganglion,heart, or muscle, and a combination thereof. In some embodiments, thecell is a neuron (optionally a dorsal root ganglion neuron), astrocyte,cardiomyocyte, or myocyte.

In some embodiments, the subject has a neurodegenerative disease,epilepsy; stroke; spinocerebellar ataxia; Canavan's disease;Metachromatic leukodystrophy; Spinal muscular atrophy; Friedreich'sataxia; X-linked centronuclear myopathy; Lysosomal storage disease;Barth syndrome; Duchenne muscular dystrophy; Wilson's disease; orCrigler-Najjar syndrome type 1. In some embodiments, theneurodegenerative disease is Parkinson's disease; Alzheimer's disease;Huntington's disease; Amyotrophic lateral sclerosis; and Multiplesclerosis.

In some embodiments, the subject has a brain cancer, and the methodincludes administering an AAV encoding an anti-cancer agent. In someembodiments, the anti-cancer agent is HSV.TK1, and the method furthercomprises administering ganciclovir.

In some embodiments, the cell is in the brain of the subject, and theAAV is administered by parenteral delivery (e.g., via intravenous,intraarterial, subcutaneous, intraperitoneal, or intramusculardelivery); intracerebral; or intrathecal delivery (e.g., via lumbarinjection, cisternal magna injection, or intraparenchymal injection).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1B depict an exemplary strategy of engineering AAV9 byinserting cell-penetrating peptides (CPPs) into its capsid. FIG. 1A is a3D model of an AAV9 virus. Individual CPP inserted into the capsidbetween amino acids 588 and 589 (VP1 numbering) will be displayed at the3-fold axis where receptor binding presumably occurs. FIG. 1B illustratethe method of individual AAV production. Three plasmids including pRC(engineered or not), pHelper and pAAV are co-transfected into HEK 293Tcells, with AAVs harvested and purified using iodixanol gradient.

FIGS. 2A-2B depict representative images of mouse brain sections andtheir quantitative analysis after intravenous administration of low-dosecandidate AAVs. Mice with mixed genetic background are used. CandidateAAVs differs in their inserted CPPs (see Table 3), but all expressnuclear red fluorescent protein (RFP) as reporter. Candidate AAVs withlow production yields are excluded for further screening. The dose ofAAV is 1×10¹⁰ vg (viral genome) per animal. Each white dot in FIG. 2Arepresents a RFP-labeled cell. In FIG. 2B, *P<0.05, vs. AAV9, ANOVA.

FIGS. 2C-2D depict representative images of mouse brain sections andtheir quantitative analysis after intravenous administration ofAAV.CPP.11 and AAV.CPP.12 in a repeat experiment. AAV.CPP.11 andAAV.CPP.12 contain CPPs BIP1 and BIP2 respectively (see Table 3). Thedoses of the AAVs are increased to 1×10¹¹ vg per animal. Candidate AAVsexpress nuclear red fluorescent protein (RFP) as reporter. Each whitedot in FIG. 2C represents a RFP-labeled cell. In FIG. 2D, *P<0.05,**P<0.01, vs. AAV9, ANOVA.

FIG. 3A depicts the optimization of the BIP targeting sequence in orderto further engineer AAV9 towards better brain transduction. BIP1 (VPALR,SEQ ID NO:1), which enables AAV9 to transduce brain more efficiently (asin AAV.CPP.11), is derived from the protein Ku70 in rats. Human, mouseand rat Ku70 proteins differ in their exact amino acid sequences. BIP2(VSALK, SEQ ID NO:2) as in AAV.CPP.12 is a “synthetic” peptide relatedto BIP1. Further engineering focuses on the VSALK sequence in the hopeof minimizing species specificity of final engineered AAV. To generatenew targeting sequence, amino acids of interest are added to the VSALKsequence, and in other cases positions of individual amino acids areswitched. All new BIP2-derived sequences are again inserted into theAAV9 capsid to generate new candidate AAVs for screening. Sequencesappearing in order are SEQ ID NOs: 69, 70, 71, 1-6, 72, 7, and 8.

FIGS. 3B-3C depict representative images of mouse brain sections andtheir quantitative analysis after intravenous administration of morecandidate AAVs. All candidate AAVs express nuclear red fluorescentprotein (RFP) as reporter. The dose of AAV is 1×10¹¹ vg per animal. Eachwhite dot in FIG. 3B represents a RFP-labeled cell. AAV.CPP.16 andAAV.CPP.21 were identified as top hits with their robust and widespreadbrain transduction. In FIG. 3C, *P<0.05, **P<0.01, ***P<0.001, vs. AAV9,ANOVA.

FIG. 3D depicts quantitative analysis of transduction efficiency in theliver after intravenous administration of candidate AAVs. Percentage oftransduced liver cells is presented. The dose of AAV is 1×10¹¹ vg peranimal. ***P<0.001, vs. AAV9, ANOVA.

FIGS. 4A-4E depict screening of selected candidate AAVs in an in vitrospheroid model of human blood-brain barrier. FIG. 4A illustrates thespheroid comprising human microvascular endothelial cells, which forms abarrier at the surface, and human pericyte and astrocytes inside thespheriod. Candidate AAVs were assessed for their ability to penetratefrom the surrounding medium into the inside of the spheroid and totransduce the cells inside. FIG. 4B-4D shows images of AAV9, AAV.CPP.16and AAV.CPP.21 treated spheroids. FIG. 4E shows relative RFP intensityof different AAV treated spheroids. ***P<0.001, vs. AAV9, ANOVA.

FIGS. 5A-5B depict representative images of brain sections and theirquantitative analysis after intravenous administration of AAV9,AAV.CPP.16 and AAV.CPP.21 in C57BL/6J inbred mice. All candidate AAVsexpress nuclear red fluorescent protein (RFP) as reporter. The dose ofAAV is 1×10¹² vg per animal. Each white dot in FIG. 5A represents aRFP-labeled cell. In FIG. 5B, *P<0.05, ***P<0.001, ANOVA.

FIGS. 6A-6B depict representative images of brain sections and theirquantitative analysis after intravenous administration of AAV9,AAV.CPP.16 and AAV.CPP.21 in BALB/cJ inbred mice. All candidate AAVsexpress nuclear red fluorescent protein (RFP) as reporter. The dose ofAAV is 1×10¹² vg per animal. Each white dot in FIG. 6A represents aRFP-labeled cell. In FIG. 6B, ***P<0.001, ANOVA.

FIGS. 7A-7B depict representative images of brain sections and theirquantitative analysis after intravenous administration of high-doseAAV.CPP.16 and AAV.CPP.21 in C57BL/6J inbred mice. Both candidate AAVsexpress nuclear red fluorescent protein (RFP) as reporter. The dose ofAAV is 4×10¹² vg per animal. Each white dot in FIG. 7A represents aRFP-labeled cell. In FIG. 7B, *P<0.05, Student test.

FIG. 8A shows AAV.CPP.16 and AAV.CPP.21 transduce adult neurons (labeledby a NeuN antibody) across multiple brain regions in mice including thecortex, midbrain and hippocampus. Transduced neurons are co-labeled byNeuN antibody and RFP. AAVs of 4×10¹² vg were administered intravenouslyin adult C57BL/6J mice (6 weeks old).

FIG. 8B depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs.AAV9 in targeting the spinal cord and motor neurons in mice. AAVs of4×10¹⁰ vg were administered intravenously into neonate mice (1 day afterbirth). Motor neurons in the ventral horn of the spinal cord werevisualized using CHAT antibody staining. Co-localization of RFP and CHATsignals suggests specific transduction of the motor neurons.

FIG. 9A depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 intargeting the heart in adult mice. AAVs of 1×10¹¹ vg were administeredintravenously in adult C57BL/6J mice (6 weeks old). Percentage ofRFP-labeled cells relative to all DAPI-stained cells is presented.*P<0.05, Student test.

FIG. 9B depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 intargeting the skeletal muscle in adult mice. AAVs of 1×10¹¹ vg wereadministered intravenously in adult C57BL/6J mice (6 weeks old).Percentage of RFP-labeled cells relative to all DAPI-stained cells ispresented. *P<0.05, Student test.

FIG. 9C depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 intargeting the dorsal root ganglion (DRG) in adult mice. AAVs of 1×10¹¹vg were administered intravenously in adult C57BL/6J mice (6 weeks old).Percentage of RFP-labeled cells relative to all DAPI-stained cells ispresented. *P<0.05, Student test.

FIG. 10A depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced abilityvs. AAV9 to transduce brain cells in primary visual cortex afterintravenous administration in non-human primates. 2×10¹³ vg/kgAAVs-CAG-AADC (as reporter gene) were injected intravenously into 3months old cynomolgus monkeys with low pre-existing neutralizingantibody. AAV-transduced cells (shown in black) were visualized usingantibody staining against AADC. Squared areas in the left panels areenlarged as in the right panels. AAV.CPP.16 transduced significantlymore cells vs. AAV9. AAV.CPP.21 also transduced more cell vs. AAV9although its effect was less evident in comparison with AAV.CPP.16.

FIG. 10B depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced abilityvs. AAV9 to transduce brain cells in parietal cortex after intravenousadministration in non-human primates. 2×10¹³ vg/kg AAVs-CAG-AADC (asreporter gene) were injected intravenously into 3 months old cynomolgusmonkeys with low pre-existing neutralizing antibody. AAV-transducedcells (shown in black) were visualized using antibody staining againstAADC. Squared areas in the left panels are enlarged as in the rightpanels. AAV.CPP.16 transduced significantly more cells vs. AAV9.AAV.CPP.21 also transduced more cell vs. AAV9 although its effect wasless evident in comparison with AAV.CPP.16.

FIG. 10C depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced abilityvs. AAV9 to transduce brain cells in thalamus after intravenousadministration in non-human primates. 2×10¹³ vg/kg AAVs-CAG-AADC (asreporter gene) were injected intravenously into 3 months old cynomolgusmonkeys with low pre-existing neutralizing antibody. AAV-transducedcells (shown in black) were visualized using antibody staining againstAADC. Squared areas in the left panels are enlarged as in the rightpanels. AAV.CPP.16 transduced significantly more cells vs. AAV9.AAV.CPP.21 also transduced more cell vs. AAV9 although its effect wasless evident in comparison with AAV.CPP.16.

FIG. 10D depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced abilityvs. AAV9 to transduce brain cells in cerebellum after intravenousadministration in non-human primates. 2×10¹³ vg/kg AAVs-CAG-AADC (asreporter gene) were injected intravenously into 3 months old cynomolgusmonkeys with low pre-existing neutralizing antibody. AAV-transducedcells (shown in black) were visualized using antibody staining againstAADC. Squared areas in the left panels are enlarged as in the rightpanels. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly morecells vs. AAV9.

FIGS. 11A-11B depict that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A.LY6A serves as a receptor for AAV.PHP.B and its variants includingAAV.PHP.eB (as in U.S. Pat. No. 9,102,949, US20170166926) and mediatesAAV.PHP.eB's robust effect in crossing the BBB in certain mouse strains(Hordeaux et al. Mol Ther 2019 27(5):912-921; Huang et al. 2019,dx.doi.org/10.1101/538421). Over-expressing mouse LY6A in cultured 293cells significantly increases binding of AAV.PHP.eB to the cell surface(FIG. 11A). On the contrary, over-expressing LY6A does not increaseviral binding for AAV9, AAV.CPP.16 or AAV.CPP.21 (FIG. 11B). Thissuggests AAV.CPP.16 or AAV.CPP.21 does not share LY6A with AAV.PHP.eB asa receptor.

FIGS. 12A-12C depict that AAV.CPP.21 can be used to systemically delivera therapeutic gene into brain tumor in a mouse mode of glioblastoma(GBM). In FIG. 12A, as in FIG. 11A, intravenously administeredAAV.CPP.21-H2BmCherry was shown to target tumor mass, especially thetumor expanding frontier. In FIG. 12B-12C, using AAV.CPP.21 tosystemically deliver the “suicide gene” HSV.TK1 results in shrinkage ofbrain tumor mass, when combined with the pro-drug ganciclovir. HSV.TK1turns the otherwise “dormant” ganciclovir into a tumor-killing drug.*P<0.05, Student test.

FIG. 13 depicts that when injected locally into adult mouse brain,AAV.CPP.21 resulted in more widespread and robust transduction of braintissue in comparison with AAV9. Intracerebral injection of AAVs (1×10¹¹vg) was performed in adult mice (>6 weeks old) and brain tissues wereharvested and examined 3 weeks after AAV injection. **P<0.01, Studenttest.

DETAILED DESCRIPTION

Difficulties associated with delivery across the BBB have hindereddevelopment of therapeutic agents to treat brain disorders includingcancer and neurodegenerative disorders. Adeno-associated virus (AAV) hasemerged as an important research and clinical tool for deliveringtherapeutic genes to the brain, spinal cord and the eye; see, e.g., U.S.Pat. Nos. 9,102,949; 9,585,971; and US20170166926. However, existingAAVs including AAV9 have either limited efficiency in crossing the BBB,or only work in some non-primate species.

Through rational design and targeted screening on the basis of knowncell-penetrating peptides (CPPs) (see, e.g., Gomez et al.,Bax-inhibiting peptides derived from Ku70 and cell-penetratingpentapeptides. Biochem. Soc. Trans. 2007; 35(Pt 4):797-801), targetingsequences have been discovered that, when engineered into the capsid ofan AAV, improved the efficiency of gene delivery to the brain by up tothree orders of magnitude. These methods were used to engineer one suchAAV vector that dramatically reduced tumor size in an animal model ofglioblastoma.

Targeting Sequences

The present methods identified a number of potential targeting peptidesthat enhance permeation through the BBB, e.g., when inserted into thecapsid of an AAV, e.g., AAV1, AAV2, AAV8, or AAV9, or when conjugated toa biological agent, e.g., an antibody or other large biomolecule, eitherchemically or via expression as a fusion protein.

In some embodiments, the targeting peptides comprise sequences of atleast 5 amino acids. In some embodiments, the amino acid sequencecomprises at least 4, e.g., 5, contiguous amino acids of the sequencesVPALR (SEQ ID NO:1) and VSALK (SEQ ID NO:2).

In some embodiments, the targeting peptides comprise a sequence of X₁ X₂X₃ X₄ X₅, wherein:

-   (i) X₁, X₂, X₃, X₄ are any four non-identical amino acids of V, A,    L, I, G, P, S, T, or M; and-   (ii) X₅ is K, R, H, D, or E (SEQ ID NO:73).

In some embodiments, the targeting peptides comprise sequences of atleast 6 amino acids. In some embodiments, the amino acid sequencecomprises at least 4, e.g., 5 or 6 contiguous amino acids of thesequences TVPALR (SEQ ID NO:3), TVSALK (SEQ ID NO:4), TVPMLK (SEQ ID NO:12) and TVPTLK (SEQ ID NO:13).

In some embodiments, the targeting peptides comprise a sequence of X₁ X₂X₃ X₄ X₅ X₆, wherein:

-   (i) X₁ is T;-   (ii) X₂, X₃, X₄, X₅ are any four non-identical amino acids of V, A,    L, I, G, P, S, T, or M; and-   (iii) X₆ is K, R, H, D, or E (SEQ ID NO:74).

In some embodiments, the targeting peptides comprise a sequence of X₁ X₂X₃ X₄ X₅ X₆, wherein:

-   (i) X₁, X₂, X₃, X₄ are any four non-identical amino acids from V, A,    L, I, G, P, S, T, or M;-   (ii) X₅ is K, R, H, D, or E; and-   (iii) X₆ is E or D (SEQ ID NO:75).

In some embodiments, the targeting peptides comprise sequences of atleast 7 amino acids. In some embodiments, the amino acid sequencecomprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of thesequences FTVSALK (SEQ ID NO:5), LTVSALK (SEQ ID NO:6), TVSALFK (SEQ IDNO:8), TVPALFR (SEQ ID NO:9), TVPMLFK (SEQ ID NO:10) and TVPTLFK (SEQ IDNO:11). In some other embodiments, the targeting peptides comprise asequence of X₁ X₂ X₃ X₄ X₅ X₆ X₇, wherein:

-   (i) X₁ is F, L, W, or Y;-   (ii) X₂ is T;-   (iii) X₃, X₄, X₅, X₆ are any four non-identical amino acids of V, A,    L, I, G, P, S, T, or M; and-   (iv) X₇ is K, R, H, D, or E (SEQ ID NO:76).

In some embodiments, the targeting peptides comprise a sequence of X₁ X₂X₃ X₄ X₅ X₆ X₇, wherein:

-   (i) X₁ is T;-   (ii) X₂, X₃, X₄, X₅ are any four non-identical amino acids of V, A,    L, I, G, P, S, T, or M;-   (iii) X₆ is K, R, H, D, or E; and-   (iv) X₇ is E or D (SEQ ID NO:77).

In some embodiments, the targeting peptides comprise a sequence of X₁ X₂X₃ X₄ X₅ X₆ X₇, wherein:

-   (i) X₁, X₂, X₃, X₄ are any four non-identical amino acids of V, A,    L, I, G, P, S, T, or M;-   (ii) X₅ is K, R, H, D, or E;-   (iii) X₆ is E or D; and-   (iv) X₇ is A or I (SEQ ID NO:78).

In some embodiments, the targeting peptides comprise a sequence of V[S/p][A/m/t/]L (SEQ ID NO:79), wherein the upper case letters arepreferred at that position. In some embodiments, the targeting peptidescomprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:80). In someembodiments, the targeting peptides comprise a sequence ofTV[S/p][A/m/t/]LK (SEQ ID NO:81). In some embodiments, the targetingpeptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO:82).

In some embodiments, the targeting peptide does not consist of VPALR(SEQ ID NO:1) or VSALK (SEQ ID NO:2).

Specific exemplary amino acid sequences that include the above mentioned5, 6, or 7-amino acid sequences are listed in Table 1.

TABLE 1 Targeting Sequences SEQ ID NO: Targeting Peptide Sequence  1.VPALR  2. VSALK  3. TVPALR  4. TVSALK  5. FTVSALK  6. LTVSALK  7.TFVSALK  8. TVSALFK  9. TVPALFR 10. TVPMLFK 11. TVPTLFK 12. TVPMLK 13.TVPTLK 14. VPMLK 15. VPTLK 16. VPMLKE 17. VPTLKD 18. VPALRD 19. VSALKE20. VSALKD 21. TAVSLK 22. TALVSK 23. TVLSAK 24. TLVSAK 25. TMVPLK 26.TMLVPK 27. TVLPMK 28. TLVPMK 29. TTVPLK 30. TTLVPK 31. TVLPTK 32. TLVPTK33. TAVPLR 34. TALVPR 35. TVLPAR 36. TLVPAR 37. TAVSLKE 38. TALVSKE 39.TVLSAKE 40. TLVSAKE 41. TMVPLKE 42. TMLVPKE 43. TVLPMKE 44. TLVPMKE 45.TTVPLKD 46. TTLVPKD 47. TVLPTKD 48. TLVPTKD 49. TAVPLRD 50. TALVPRD 51.TVLPARD 52. TLVPARD 53. TAVSLFK 54. TALVSFK 55. TVLSAFK 56. TLVSAFK 57.TMVPLFK 58. TMLVPFK 59. TVLPMFK 60. TLVPMFK 61. TTVPLFK 62. TTLVPFK 63.TVLPTFK 64. TLVPTFK 65. TAVPLFR 66. TALVPFR 67. TVLPAFR 68. TLVPAFR

Targeting peptides including reversed sequences can also be used, e.g.,KLASVT (SEQ ID NO:83) and KFLASVT (SEQ ID NO:84).

Targeting peptides disclosed herein can be modified according to themethods known in the art for producing peptidomimetics. See, e.g., Qvitet al., Drug Discov Today. 2017 February; 22(2): 454-462; Farhadi andHashemian, Drug Des Devel Ther. 2018; 12: 1239-1254; Avan et al., Chem.Soc. Rev., 2014,43, 3575-3594; Pathak, et al., Indo American Journal ofPharmaceutical Research, 2015. 8; Kazmierski, W. M., ed.,Peptidomimetics Protocols, Human Press (Totowa N.J. 1998); Goodman etal., eds., Houben-Weyl Methods of Organic Chemistry: Synthesis ofPeptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo etal., J. Biol. Chem., 278:45746 (2003). In some cases, these modifiedpeptidomimetic versions of the peptides and fragments disclosed hereinexhibit enhanced stability in vivo, relative to the non-peptidomimeticpeptides.

Methods for creating a peptidomimetic include substituting one or more,e.g., all, of the amino acids in a peptide sequence with D-amino acidenantiomers. Such sequences are referred to herein as “retro” sequences.In another method, the N-terminal to C-terminal order of the amino acidresidues is reversed, such that the order of amino acid residues fromthe N-terminus to the C-terminus of the original peptide becomes theorder of amino acid residues from the C-terminus to the N-terminus inthe modified peptidomimetic. Such sequences can be referred to as“inverso” sequences.

Peptidomimetics can be both the retro and inverso versions, i.e., the“retro-inverso” version of a peptide disclosed herein. The newpeptidomimetics can be composed of D-amino acids arranged so that theorder of amino acid residues from the N-terminus to the C-terminus inthe peptidomimetic corresponds to the order of amino acid residues fromthe C-terminus to the N-terminus in the original peptide.

Other methods for making a peptidomimetic include replacing one or moreamino acid residues in a peptide with a chemically distinct butrecognized functional analog of the amino acid, i.e., an artificialamino acid analog. Artificial amino acid analogs include β-amino acids,β-substituted β-amino acids (“β³-amino acids”), phosphorous analogs ofamino acids, such as ∀-amino phosphonic acids and V-amino phosphinicacids, and amino acids having non-peptide linkages. Artificial aminoacids can be used to create peptidomimetics, such as peptoid oligomers(e.g., peptoid amide or ester analogues), β-peptides, cyclic peptides,oligourea or oligocarbamate peptides; or heterocyclic ring molecules.Exemplary retro-inverso targeting peptidomimetics include KLASVT andKFLASVT, wherein the sequences include all D-amino acids. Thesesequences can be modified, e.g., by biotinylation of the amino terminusand amidation of the carboxy terminus.

AAVs

Viral vectors for use in the present methods and compositions includerecombinant retroviruses, adenovirus, adeno-associated virus,alphavirus, and lentivirus, comprising the targeting peptides describedherein and optionally a transgene for expression in a target tissue.

A preferred viral vector system useful for delivery of nucleic acids inthe present methods is the adeno-associated virus (AAV). AAV is a tinynon-enveloped virus having a 25 nm capsid. No disease is known or hasbeen shown to be associated with the wild type virus. AAV has asingle-stranded DNA (ssDNA) genome. AAV has been shown to exhibitlong-term episomal transgene expression, and AAV has demonstratedexcellent transgene expression in the brain, particularly in neurons.Vectors containing as little as 300 base pairs of AAV can be packagedand can integrate. Space for exogenous DNA is limited to about 4.7 kb.An AAV vector such as that described in Tratschin et al., Mol. Cell.Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. Avariety of nucleic acids have been introduced into different cell typesusing AAV vectors (see for example Hermonat et al., Proc. Natl. Acad.Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol.4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988);Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J.Biol. Chem. 268:3781-3790 (1993). There are numerous alternative AAVvariants (over 100 have been cloned), and AAV variants have beenidentified based on desirable characteristics. In some embodiments, theAAV is AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10,rh.39, rh.43 or CSp3; for CNS use, in some embodiments the AAV is AAV1,AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As one example, AAV9 has beenshown to somewhat efficiently cross the blood-brain barrier. Using thepresent methods, the AAV capsid can be genetically engineered toincrease permeation across the BBB, or into a specific tissue, byinsertion of a targeting sequence as described herein into the capsidprotein, e.g., into the AAV9 capsid protein VP1 between amino acids 588and 589.

An exemplary wild type AAV9 capsid protein VP1 (Q6JC40-1) sequence is asfollows:

(SEQ ID NO: 85)         10         20         30         40MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD        50         60         70         80NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD        90        100        110        120QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ       130        140        150        160AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG       170        180        190        200KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS       210        220        230        240LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI       250        260        270        280TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP       290        300        310        320WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI       330        340        350        360QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH       370        380        390        400EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF       410        420        430        440PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI       450        460        470        480DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP       490        500        510        520GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP       530        540        550        560GPAMASHKEG EDRFFPLSGS LIFGKQGTGR DNVDADKVMI       570        580        590        600TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG       610        620        630        640ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM       650        660        670        680KHPPPQILIK NTPVPADPPT AFNKDKLNSF ITQYSTGQVS       690        700        710        720VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV        730 YSEPRPIGTR YLTRNL

Thus provided herein are AAV that include one or more of the targetingpeptide sequences described herein, e.g., an AAV comprising a capsidprotein comprising a targeting sequence described herein, e.g., a capsidprotein comprising SEQ ID NO:1 wherein a targeting peptide sequence hasbeen inserted into the sequence, e.g., between amino acids 588 and 589.

In some embodiments, the AAV also includes a transgene sequence (i.e., aheterologous sequence), e.g., a transgene encoding a therapeutic agent,e.g., as described herein or as known in the art, or a reporter protein,e.g., a fluorescent protein, an enzyme that catalyzes a reactionyielding a detectable product, or a cell surface antigen. The transgeneis preferably linked to sequences that promote/drive expression of thetransgene in the target tissue.

Exemplary transgenes for use as therapeutics include neuronal apoptosisinhibitory protein (NAIP), nerve growth factor (NGF), glial-derivedgrowth factor (GDNF), brain-derived growth factor (BDNF), ciliaryneurotrophic factor (CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase(GTPCH), amino acid decorboxylase (AADC), aspartoacylase (ASPA), bloodfactors, such as β-globin, hemoglobin, tissue plasminogen activator, andcoagulation factors; colony stimulating factors (CSF); interleukins,such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.;growth factors, such as keratinocyte growth factor (KGF), stem cellfactor (SCF), fibroblast growth factor (FGF, such as basic FGF andacidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors(IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF),growth differentiation factor-9 (GDF-9), hepatoma derived growth factor(HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins,platelet-derived growth factor (PDGF), thrombopoietin (TPO),transforming growth factor alpha (TGF-α), transforming growth factorbeta (TGF-β), and the like; soluble receptors, such as soluble TNF-αreceptors, soluble VEGF receptors, soluble interleukin receptors (e.g.,soluble IL-1 receptors and soluble type II IL-1 receptors), solublegamma/delta T cell receptors, ligand-binding fragments of a solublereceptor, and the like; enzymes, such as α-glucosidase, imiglucarase,β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissueplasminogen activator; chemokines, such as IP-10, monokine induced byinterferon-gamma (Mig), Groa/IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4,and the like; angiogenic agents, such as vascular endothelial growthfactors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforminggrowth factor-beta, basic fibroblast growth factor, glioma-derivedgrowth factor, angiogenin, angiogenin-2; and the like; anti-angiogenicagents, such as a soluble VEGF receptor; protein vaccine; neuroactivepeptides, such as nerve growth factor (NGF), bradykinin,cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasinghormone, beta-endorphin, enkephalin, substance P, somatostatin,prolactin, galanin, growth hormone-releasing hormone, bombesin,dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y,luteinizing hormone, calcitonin, insulin, glucagons, vasopressin,angiotensin II, thyrotropin-releasing hormone, vasoactive intestinalpeptide, a sleep peptide, and the like; thrombolytic agents; atrialnatriuretic peptide; relaxin; glial fibrillary acidic protein; folliclestimulating hormone (FSH); human alpha-1 antitryp sin; leukemiainhibitory factor (LIF); transforming growth factors (TGFs); tissuefactors, luteinizing hormone; macrophage activating factors; tumornecrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growthfactor; tissue inhibitors of metalloproteinases; vasoactive intestinalpeptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptorantagonists; and the like. Some other examples of protein of interestinclude ciliary neurotrophic factor (CNTF); neurotrophins 3 and 4/5(NT-3 and 4/5); glial cell derived neurotrophic factor (GDNF); aromaticamino acid decarboxylase (AADC); hemophilia related clotting proteins,such as Factor VIII, Factor IX, Factor X; dystrophin or nini-dystrophin;lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storagedisease-related enzymes, such as glucose-6-phosphatase, acid maltase,glycogen debranching enzyme, muscle glycogen phosphorylase, liverglycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase(e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase,and glycogen synthase; lysosomal enzymes (e.g.,beta-N-acetylhexosaminidase A); and any variants thereof.

The transgene can also encode an antibody, e.g., an immune checkpointinhibitory antibody, e.g., to PD-L1, PD-1, CTLA-4 (CytotoxicT-Lymphocyte-Associated Protein-4; CD152); LAG-3 (Lymphocyte ActivationGene 3; CD223); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3;HAVCR2); TIGIT (T-cell Immunoreceptor with Ig and ITIM domains); B7-H3(CD276); VSIR (V-set immunoregulatory receptor, aka VISTA, B7H5,C10orf54); BTLA 30 (B- and T-Lymphocyte Attenuator, CD272); GARP(Glycoprotein A Repetitions; Predominant; PVRIG (PVR relatedimmunoglobulin domain containing); or VTCN1 (Vset domain containing Tcell activation inhibitor 1, aka B7-H4).

Other transgenes can include small or inhibitory nucleic acids thatalter/reduce expression of a target gene, e.g., siRNA, shRNA, miRNA,antisense oligos, or long non-coding RNAs that alter gene expression(see, e.g., WO2012087983 and US20140142160), or CRISPR Cas9/cas12a andguide RNAs.

The virus can also include one or more sequences that promote expressionof a transgene, e.g., one or more promoter sequences; enhancersequences, e.g., 5′ untranslated region (UTR) or a 3′ UTR; apolyadenylation site; and/or insulator sequences. In some embodiments,the promoter is a brain tissue specific promoter, e.g., aneuron-specific or glia-specific promoter. In certain embodiments, thepromoter is a promoter of a gene selected to from: neuronal nuclei(NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatouspolyposis coli (APC), ionized calcium-binding adapter molecule 1(Iba-1), synapsin I (SYN), calcium/calmodulin-dependent protein kinaseII, tubulin alpha I, neuron-specific enolase and platelet-derived growthfactor beta chain. In some embodiments, the promoter is a pan-cell typepromoter, e.g., cytomegalovirus (CMV), beta glucuronidase, (GUSB),ubiquitin C (UBC), or rous sarcoma virus (RSV) promoter. The woodchuckhepatitis virus posttranscriptional response element (WPRE) can also beused.

In some embodiments, the AAV also has one or more additional mutationsthat increase delivery to the target tissue, e.g., the CNS, or thatreduce off-tissue targeting, e.g., mutations that decrease liverdelivery when CNS, heart, or muscle delivery is intended (e.g., asdescribed in Pulicherla et al. (2011) Mol Ther 19:1070-1078); or theaddition of other targeting peptides, e.g., as described in Chen et al.(2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 orU.S. Pat. Nos. 9,102,949; 9,585,971; and US20170166926. See also Grayand Samulski (2011) “Vector design and considerations for CNSapplications,” in Gene Vector Design and Application to Treat NervousSystem Disorders ed. Glorioso J., editor. (Washington, D.C.: Society forNeuroscience;) 1-9, available atsfn.org/˜/media/SfN/Documents/Short%20Courses/2011%20Short%20Course%20I/2011_SC1_Gray.ashx.

Targeting Peptides as Tags/Fusions

The targeting peptides described herein can also be used to increasepermeation of other (heterologous) molecules across the BBB, e.g., byconjugation to the molecule, or by expression as part of a fusionprotein, e.g., with an antibody or other large biomolecule. These caninclude genome editing proteins or complexes (e.g., TALEs, ZFNs, Baseeditors, and CRISPR RNPs comprising a gene editing protein such as Cas9or Cas12a, fused to a peptide described herein (e.g., at the N terminus,C terminus, or internally) and a guide RNA), in addition to therapeuticagents or reporters as described herein as well as those listed in Table2. The fusions/complexes do not comprise any other sequences from Ku70,e.g., comprise heterologous non-Ku70 sequences, and are not present innature.

In some embodiments, targeting sequences used as part of a non-AAVfusion protein do not comprise or consist of VPALR (SEQ ID NO:1) orVSALK (SEQ ID NO:2).

Methods of Use

The methods and compositions described herein can be used to deliver anycomposition, e.g., a sequence of interest to a tissue, e.g., to thecentral nervous system (brain), heart, muscle, or dorsal root ganglionor spinal cord (peripheral nervous system). In some embodiments, themethods include delivery to specific brain regions, e.g., cortex,cerebellum, hippocampus, substantia nigra, amygdala. In someembodiments, the methods include delivery to neurons, astrocytes, glialcells, or cardiomyocytes.

In some embodiments, the methods and compositions, e.g., AAVs, are usedto deliver a nucleic acid sequence to a subject who has a disease, e.g.,a disease of the CNS; see, e.g., U.S. Pat. Nos. 9,102,949; 9,585,971;and US20170166926. In some embodiments, the subject has a conditionlisted in Table 2; in some embodiments, the vectors are used to delivera therapeutic agent listed in Table 2 for treating the correspondingdisease listed in Table 2. The therapeutic agent can be delivered as anucleic acid, e.g. via a viral vector, wherein the nucleic acid encodesa therapeutic protein or other nucleic acid such as an antisense oligo,siRNA, shRNA, and so on; or as a fusion protein/complex with a targetingpeptide as described herein.

TABLE 2 Diseases Examples of diseases Tissue targeted Therapeutic agentParkinson's disease CNS GDNF, AADC Alzheimer's disease CNS Tau antibody,APP antibody Huntington's disease CNS miRNA targeting HTT Amyotrophiclateral CNS shRNA targeting SOD sclerosis Multiple sclerosis CNSIFN-beta Epilepsy CNS Neuropeptide Y Stroke CNS IGF-1, osteopontin Braincancer CNS HSV.TK1, PD-1/PD- L1 antibody Spinocerebellar ataxia CNS RNAitargeting ataxin Canavan disease CNS ASPA Metachromatic Nervous systemsARSA, PSAP leukodystrophy Spinal muscular atrophy Neuromuscular systemSMN1 Friedreich's ataxia Nervous systems, heart Frataxin X-linkedmyotubular Neuromuscular system MTM1 myopathy Pompe disease Lysosome(global GAA including CNS) Barth syndrome Heart, muscle TAZ Duchennemuscular Muscle dystrophin dystrophy Wilson's disease Brain, liver ATP7BCrigler-Najjar syndrome Liver UGT1A1 type 1

In some embodiments, the compositions and methods are used to treatbrain cancer. Brain cancers include gliomas (e.g., glioblastomamultiforme (GBM)), metastases (e.g., from lung, breast, melanoma, orcolon cancer), meningiomas, pituitary adenomas, and acoustic neuromas.The compositions include a targeting peptide linked to an anticanceragent, e.g., a “suicide gene” that induces apoptosis in a target cell(e.g., HSV.TK1, Cytosine Deaminase (CD) from Herpes simplex virus orEscherichia coli, or Escherichia coli purine nucleoside phosphorylase(PNP)/fludarabine; see Krohne et al., Hepatology. 2001 September;34(3):511-8; Dey and Evans, “Suicide Gene Therapy by Herpes SimplexVirus-1 Thymidine Kinase (HSV-TK)” (2011) DOI: 10.5772/18544) an immunecheckpoint inhibitory antibody as known in the art or described herein.For example, an AAV vector comprising a targeting peptide as describedherein can be used to deliver the “suicide gene” HSV.TK1 to a braintumor. HSV.TK1 turns the otherwise “dormant” ganciclovir into atumor-killing drug. Thus the methods can include systemically, e.g.,intravenously, administering an AAV (e.g., AAV9) comprising a targetingpeptide as described herein and encoding HSV.TK1, and the pro-drugganciclovir, to a subject who has been diagnosed with brain cancer.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the use of pharmaceuticalcompositions comprising the targeting peptides as an active ingredient.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intraarterial,subcutaneous, intraperitoneal intramuscular or injection or infusionadministration. Delivery can thus be systemic or localized.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, the therapeutic compounds are prepared with carriersthat will protect the therapeutic compounds against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Such formulations can be prepared using standardtechniques, or obtained commercially, e.g., from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to selected cells with monoclonal antibodies to cellularantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration. Forexample, the compositions comprising an AAV comprising a targetingpeptide as described herein and a nucleic acid encoding HSV.TK1 can beprovided in a kit with ganciclovir.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples below.

1. Generation of Capsid Variants

To generate the capsid variant plasmids, DNA fragments that encode thecell-penetrating peptides (Table 3) were synthesized (GenScript), andinserted into the backbone of the AAV9 Rep-cap plasmid (pRC9) betweenamino acid position 588 and 589 (VP1 amino acid numbering), usingCloneEZ seamless cloning technology (GenScript). CPPs BIP1 (VPALR, SEQID NO:1) and BIP2 (VSALK, SEQ ID NO:2), as well as their derivativessuch as TVSALK (SEQ ID NO:4) in AAV.CPP.16 and TVSALFK (SEQ ID NO:8) inAAV.CPP.21, are derived from the Ku70 proteins, of which the sequencesare provided as below:

Human Ku70 MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPF 60 Mouse Ku70MSEWESYYKTEGEEEEEE--EESPDTGGEYKYSGRDSLIFLVDASRAMFESQGEDELTPF  58Rat Ku70 MSEWESYYKTEGEEEEEE--EQSPDTNGEYKYSGRDSLIFLVDASRLMFESQGEDELTPF 58 Human Ku70DMSIQCIQSVYISKIISSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELD 120Mouse Ku70 DMSIQCIQSVYTSKIISSDRDLLAVVYYGTEKDKNSVNFKNIYVLQDLDNPGAKRVLELD118 Rat Ku70DMSIQCIQSVYTSKIISSDRDLLAVVFYGTEKDKNSVNFKSIYVLQDLDNPGAKRVLELD 118Human Ku70 QFKGQQGQKRFQDMMGHGSDYSLSEVLWVCANLFSDVQFKMSHKRIMLFTNEDNPHGNDS180 Mouse Ku70QFKGQQGKKHFRDTVGHGSDYSLSEVLWVCANLFSDVQLKMSHKRIMLFTNEDDPHGRDS 178Rat Ku70 RFKGQQGKKHFRDTIGHGSDYSLSEVLWVCANLFSDVQFKMSHKRIMLFTNEDDPHGNDS178 Human Ku70AKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDIISIAEDEDLRVHFEESSKLE 240Mouse Ku70 AKASRARTKASDLRDTGIFLDLMHLKKPGGFDVSVFYRDIITTAEDEDLGVHFEESSKLE238 Rat Ku70AKASRARTKASDLRDTGIFLDLMHLKKRGGFDVSLFYRDIISIAEDEDLGVHFEESSKLE 238Human Ku70 DLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKALKPPPIKLYRETNEPVKTKT300 Mouse Ku70DLLRKVRAKETKKRVLSRLKFKLGEDVVLMVGIYNLVQKANKPFPVRLYRETNEPVKTKT 298Rat Ka70 DLLRKVRAKETKKRVLSRLKFKLGKDVALMVGVYNLVQKANKPFPVRLYRETNEPVKTKT298 Human Ku70RTFNTSTGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFDDPGLMLMGFKPLVLLKKHH 360Mouse KU70 RTFNVNTGSLLLPSDTKRSLTYGTRQIVLEKEETEELKRFDEPGLILMGFKPTVMLKKQH358 Rat Ka70RTFNVNTGSLLLPSDTKRSLTFGTRQIVLEKEETEELKRFDEPGLILMGFKPMVMLKNHH 358Human Ku70 YLRPSLFVYPEESLVIGSSTLFSALLIKCLEKEVAALCRYTPRRNIPPYFVALVPQEEEL420 Mouse Ku70YLRPSLFVYPEESLVSGSSTLFSALLTKCVEKEVIAVCRYTPRKNVSPYFVALVPQEEEL 418Rat Ku70 YLRPSLFLYPEESLVNGSSTLFSALLTKCVEKEVIAVCRYTARKNVSPYFVALVPQEEEL418 Human Ku70DDQKIQVTPPGFQLVFLPFADDKRKMPFTEKIMATPEQVGKMKAIVEKLRFTYRSDSFEN 480Mouse Ku70 DDQNIQVTPGGFQLVFLPYADDKRKVPFTEKVTANQEQIDKMKAIVQKLRFTYRSDSFEN478 Rat Ku70DDQNIQVTPAGFQLVFLPYADDKRKVPFTEKVMANPEQIDKMKAIVQKLRFTYRSDSFEN 478Human Ku70 PVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRLGSLVDEFKELVYPPDYNPEGKV540 Mouse Ku70PVLQQHFRNLEALALDMMESEQVVDLTLPKVEAIKKRLGSLADEFKELVYPPGYNPEGKV 538Rat Ku70 PVLQQHFRNLEALALDMMESEQVVDLTLPKVEAIKKRLGSLADEFKELVYPPGYNPEGKI538 Human Ku70TKRKHDNEGSGSKRPKVEYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKSGLKKQELL 600Mouse Ku70 AKRKQDDEGSTSKKPKVELSEEELKAHFRKGTLGKLTVPTLKDICKAHGLKSGPKKQELL598 Rat Ku70AKRKADNEGSASKKPKVELSEEELKDLFAKGTLGKLTVPALRDICKAYGLKSGPKKQELL 598Human Ku70 EALTKHFQD- 609 (SEQ ID NO: 86) Mouse Ku70 DALIRHLEKN608 (SEQ ID NO: 87) Rat Ku70 EALSRHLEKN 608 (SEQ ID NO: 88)

In addition, VP1 protein sequences for AAV9, AAV.CPP.16 and AAV.CPP.21are provided as below:

AAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD  60A6V.CPP16 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60 AAV.CPP21MAADGYLPDWLEDNLSEGIREWWALKPGAPQKKANQQHQDNARGLVLPGYKYLGPGNGLD  60 AAV9KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ 120A6V.CPP16 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ120 AAV.CPP21KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ 120 AAV9AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180A6V.CPP16 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE180 AAV.CPP21AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180 AAV9SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240A6V.CPP16 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI240 AAV.CPP21SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240 AAV9TTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300A6V.CPP16 TTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR300 AAV.CPP21TTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300 AAV9LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360A6V.CPP16 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH360 AAV.CPP21LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360 AAV9EGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420A6V.CPP16 EGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV420 AAV.CPP21EGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420 AAV9PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480A6V.CPP16 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP480 AAV.CPP21PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480 AAV9GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540A6V.CPP16 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS540 AAV.CPP21GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540 AAV9LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ-------AQAQT 593A6V.CPP16 LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTVSAL-KAQAQT599 AAV.CPP21LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTVSALFKAQAQT 600 AAV9GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP 653A6V.CPP16 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP659 AAV.CPP21GWVQNGQGLPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP 660 AAV9VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSSNVEF 713A6V.CPP16 VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEF719 AAV.CPP21VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEF 720 AAV9AVNTEGVYSEPRPIGTRYLTRNL 736 (SEQ ID NO: 85) A6V.CPP16AVNTEGVYSEPRPIGTRYLTRNL 742 (SEQ ID NO: 89) AAV.CPP21AVNTEGVYSEPRPIGTRYLTRNL 743 (SEQ ID NO: 90)

2. Recombinant AAV Production

Recombinant AAVs were packaged using standard three-plasmidco-transfection protocol (pRC plasmid, pHelper plasmid and pAAVplasmid). pRC9 (or its variant), pHelper and pAAV carrying a transgene(e.g. nucleus-directed RFP H2B-mCherry driven by an ubiquitous EFlapromoter) were co-transfected into HEK 293T cells using polyethylenimine(PEI, Polysciences). rAAVs vectors were collected from serum-free medium72 h and 120 h post transfection and from cell at 120 h posttransfection. AAV particles in the medium were concentrated using aPEG-precipitation method with 8% PEG-8000 (wt/vol). Cell pelletscontaining viral particles were resuspended and lysed throughsonication. Combined viral vectors from PEG-precipitation and celllysates were treated with DNase and RNase at 37□ for 30 mins and thenpurified by iodixanol gradient (15%, 25%, 40% and 60%) withultracentrifugation (VTi 50 rotor, 40,000 r.p.m, 18□, 1 h). rAAVs werethen concentrated using Millipore Amicon filter unit (UFC910008, 100KMWCO) and formulated in Dulbecco's phosphate buffered saline (PBS)containing 0.001% Pluronic F68 (Gibco).

3. AAV Titering

Virus titer was determined by measuring DNase-resistant genome copiesusing quantitative PCR. pAAV-CAG-GFP was digested with PVUII(NEB) togenerate free ends for the plasmid ITRs, and was used for generating astandard curve. Virus samples were incubated with DNase I to eliminatecontaminating DNA, followed by sodium hydroxide treatment to dissolvethe viral capsid and to release the viral genome. Quantitative PCR wasperformed using an ITR Forward primer 5′-GGAACCCCTAGTGATGGAGTT (SEQ IDNO:91) and an ITR Reverse primer 5′-CGGCCTCAGTGAGCGA (SEQ ID NO: 92).Vector titers were normalized to the rAAV-2 reference standard materials(RSMs, ATCC, cat No: VR-1616, Manassas, Va.).

4. Administration of AAV in Mice

For intravenous administration, AAV diluted in sterile saline (0.2 ml)was administered through tail vein injection in adult mice (over 6 weeksof age). Animals then survived for three weeks before being euthanizedfor tissue harvesting. For intracerebral injection, AAV diluted in PBS(10 ul) was injected using a Hamilton syringe with coordinates frombregma: 1.0 mm right, 0.3 backward, 2.6 mm deep. All animal studies wereperformed in an AAALAC-accredited facility with IACUC approval.

5. Mouse Tissue Processing

Anesthetized animals were transcardially perfused with cold phosphatebuffered saline (PBS) followed by 4% paraformaldehyde (PFA). Tissueswere post-fixed in 4% PFA overnight, and then immersed in 30% sucrosesolutions for two days prior to embedding and snap-freezing in OCT.Typically, 80 um thick brain sections were cut for imaging of nativefluorescence, 40 um thick brain sections for IHC.

6. In Vitro Human BBB Spheroid Model

Hot 1% agarose (w/v, 50 ul) was added in a 96-well plate tocool/solidify. Primary human astrocytes (Lonza Bioscience), human brainmicrovascular pericytes (HBVP, ScienCell Research Laboratories) andhuman cerebral microvascular endothelial cells (hCMEC/D3; Cedarlane)were then seeded onto the agarose gel in a 1:1:1 ratio (1500 cells ofeach type). Cells were cultured at 37□ in a 5% CO2 incubator for 48-72hours to allow for spontaneous assembly of multicellular BBB spheroids.A multicellular barrier was reported to form at the periphery of thespheroid, mimicking the BBB. AAVs-H2B-mCherry were added to the culturemedium, and 4 days later all spheroids were fixed using 4% PFA,transferred into a Nunc Lab-Tek II thin-glass 8-well chamberedcoverglass (Thermo Scientific), and imaged using a Zeiss LSM710 confocalmicroscope. The intensity of RFP signal inside the spheroids wasexamined and used as a “read-out”.

7. AAV Administration in Non-Human Primate (NHP)

All NHP studies were performed by a CRO in an AAALAC-accredited facilitywith IACUC approval. Cynomolgus monkeys were pre-screened for little orno pre-existing neutralizing antibody against AAV9 (titer of <1:5). AAVdiluted in PBS/0.001% F68 was injected intravenously (via cephalic veinor femoral vein) using a peristaltic pump. 3 weeks later, animals weresubject to transcardial perfusion with PBS, followed by 4% PFA. Tissueswere then collected and processed for paraffin embedding and sectioning.

8. Immunohistochemistry

Floating staining was performed for mouse tissue sections with primaryantibodies diluted in PBS containing 10% donkey serum and 2% TritonX-100. Primary antibodies used include: chicken anti-GFP (1:1000);rabbit anti-RFP (1:1000); mouse anti-NeuN (1:500); rat anti-GFAP(1:500); Goat anti-GFAP (1:500); mouse anti-CD31 (1:500). Secondaryantibodies conjugated to fluorophores of Alexa Fluor 488, Alexa Fluor555 or Alexa Fluor 647 were applied against the primary antibody's hostspecies at a dilution of 1:200.

For paraffin sections of NHP tissue, DAB staining was performed tovisualize cells transduced by AAV-AADC. Rabbit anti-AADC antibody(1:500, Millipore) was used as primary antibody.

9. AAV Binding Assay

HEK293T cells were cultured at 37□ in a 5% CO2 incubator. One day afterseeding of HEK293T cells in a 24-well plate at a density of 250,000cells per well, a cDNA plasmid of LY6A was transiently transfected intothe cells using a transfection mixture of 200 ul DMEM (31053028; Gibco),1 ug DNA plasmid and 3 ug of PEI. 48 hours post transfection, cells wereplaced on ice to chill down for 10 mins. The medium was then changedwith 500 ul ice-cold serum-free DMEM medium containing rAAVs-mCherry atMOI of 10000. After incubating on ice for one hour, cells withpresumably AAVs bound to their surface were washed with cold PBS forthree times and were then subject to genomic DNA isolation. Cell-bindingviral particles were quantified by using qPCR with primers specific tomCherry and normalized to HEK293T genomes using human GCG as reference.

10. Mouse Model of Glioblastoma

All experiments were performed in compliance with protocols approved bythe Animal Care and Use Committees (IACUC) at the Brigham and Women'sHospital and Harvard Medical School. Syngeneic immuno-competent C57BL/6female mice weighing 20+/−1 g (Envigo) were used. GL261-Luc (100,000mouse glioblastoma cells) resuspended in 2 μL phosphate buffered saline(PBS) was injected intracranially using 10 μl syringe with a 26-gaugeneedle (80075; Hamilton). A stereotactic frame was used to locate theimplantation site (coordinates from bregma in mm: 2 right, 0.5 forward,at a depth of 3.5 into cortex). 7 days later, 200 ul AAV-HSV-TK1 (1E+12viral genomes, IV) was administered once and ganciclovir (50 mg/kg) wasadministered daily for 10 days.

Example 1. Modification of AAV9 Capsid

To identify peptide sequences that would enhance permeation of abiomolecule or virus across the blood brain barrier an AAV peptidedisplay technique was used. Individual cell-penetrating peptides, aslisted in Table 3, were inserted into the AAV9 capsid between aminoacids 588 and 589 (VP1 numbering) as illustrated in FIG. 1A. Theinsertion was carried out by modifying the RC plasmid, one of the threeplasmids co-transfected for AAV packaging; FIG. 1B shows an exemplaryschematic of the experiments. Individual AAV variants were produced andscreened separately. See Materials and Methods #1-3 for more details.

TABLE 3 No. of Name of Amino CPP Viral AVV CPP insertacid sequence of CPP # residues titer Initial AAV9 N/A N/A N/A Normalscreen- AAV.CPP.1 SynB1 RGGRLSYSRRRFSTSTGR  93 18 Low ing AAV.CPP.2 L-2HARIKPTFRRLKWKY  94 20 Low KGKFW AAV.CPP.3 PreS2-TLM PLSSIFSRIGDP  95 12Low AAV.CPP.4 Transportan AGYLLGK1NLKALAA  96 21 Low 10 LAKKIL AAV.CPP.5SAP VRLPPPVRLPPPVRLPPP  97 18 Normal AAV.CPP.6 SAP(E) VELPPPVELPPPVELPPP 98 18 Normal AAV.CPP.7 SVM3 KGTYKKKLMRIPLKGT  99 16 Low AAV.CPP.8(PPR)3 PPRPPRPPR 100  9 Normal AAV.CPP.9 (PPR)5 PPRPPRPPRPPRPPR 101 15Low AAV.CPP.10 Polyarginine RRRRRRRR 102  8 Low AAV.CPP.11 Bip1 VPALR  1  5 Normal AAV.CPP.12 Bip2 VSALK   2  5 Normal AAV.CPP.13 DPV15LRRERQSRLRRERQSR 103 16 NA AAV.CPP.14 HIV-1 Tat RKKRRQRRR 104  9 NAFollow- AAV.CPP.15 Bip1.1 TVPALR (Rat)   3  6 Normal up AAV.CPP.16Bip2.1 TVSALK (Syn)   4  6 Normal screen- AAV.CPP.17 Bip2.2FTVSALK (Syn)   5  7 Normal ing AAV.CPP.18 Bip2.3 LTVSALK (Syn)   6  7Normal AAV.CPP.19 Bip2.4 KFTVSALK (Syn)  72  8 Normal AAV.CPP.20 Bip2.5TFVSALK (Syn)   7  7 Normal AAV.CPP.21 Bip2.6 TVSALFK (Syn)   8  7Normal AAV.CPP.22 Bip2.6Rat TVPALFR (Rat)   9  7 Normal #, SEQ ID NO:Syn, synthetic

Example 2. First Round of In Vivo Screening

AAVs expressing nuclear RFP (H2B-RFP) were injected intravenously inadult mice with mixed C57BL/6 and BALB/c genetic background. 3 weekslater, brain tissues were harvested and sectioned to reveal RFP-labelledcells (white dots in FIGS. 2A and 2C, quantified in FIGS. 2B and 2D,respectively). CPPs BIP1 and BIP2 were inserted into the capsids ofAAV.CPP.11 and AAV.CPP.12, respectively. See Materials and Methods #4-5for more details.

Example 3. Optimization of Modified AAV9 Capsids

AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIPtargeting sequences. BIP inserts were derived from the protein Ku70 (SeeFIG. 3A and Material/Methods #1 for full sequence). The BIP sequenceVSALK, which is of “synthetic” origin, was chosen as a study focus tominimize potential species specificity of engineered AAV vectors. AAVswere produced and tested separately for brain transduction efficiency ascompared with AAV9 (see FIGS. 3B-C). Percentages of cell transduction inthe mouse liver 3 weeks after IV injection of some AAV variantsdelivering the reporter gene RFP are shown in FIG. 3D. See Materials andMethods #1-5 for more details.

Example 4. In Vitro Model—BBB Permeation Screening

Some of the AAV variants were screened for the ability to cross thehuman BBB using an in vitro spheroid BBB model. The spheroid containshuman microvascular endothelial cells, which form a barrier at thesurface, and human pericytes and astrocytes. AAVs carrying nuclear RFPas reporter were assessed for their ability to penetrate from thesurrounding medium into the inside of the spheroid and to transduce thecells inside. FIG. 4A shows an experimental schematic. FIGS. 4B-D showresults for wt AAV9, AAV.CPP.16, and AAV.CPP.21, respectively, those andother peptides are quantified in FIG. 4E. In this model, peptides 11,15, 16, and 21 produced the greatest permeation into the spheroids. SeeMaterials and Methods #6 for more details.

Example 5. In Vivo BBB Permeation Screening

AAV.CPP.16 and AAV.CPP.21 were selected for further evaluation in an invivo model, in experiments performed as described above for Example 2.All AAVs carried nuclear RFP as reporter. Both showed enhanced abilityvs. AAV9 to transduce brain cells after intravenous administration inC57BL/6J adult mice (white dots in brain sections in FIG. 5A, quantifiedin FIG. 5B) and in BALB/c adult mice (white dots in brain sections inFIG. 6A, quantified in FIG. 6B).

High doses of AAV.CPP.16 and AAV.CPP.21 (4×10¹² vg per mouse,administered IV) resulted in widespread brain transduction in mice. BothAAVs carried nuclear RFP as reporter (white dots in brain sections inFIG. 7A, quantified in FIG. 7B).

Example 6. In Vivo Distribution of Modified AAVs

As shown in FIG. 8A, AAV.CPP.16 and AAV.CPP.21 preferentially targetedneurons (labeled by a NeuN antibody) across multiple brain regions inmice including the cortex, midbrain and hippocampus. Both AAVs carriednuclear RFP as a reporter.

AAV.CPP.16 and AAV.CPP.21 also showed enhanced ability vs. AAV9 intargeting the spinal cord and motor neurons in mice. All AAVs carrynuclear RFP as reporter and were administered intravenously into neonatemice (4×10¹⁰ vg). Motor neurons were visualized using CHAT antibodystaining. Co-localization of RFP and CHAT signals in FIG. 8B suggestedspecific transduction of the motor neurons.

The relative abilities of AAV-CAG-H2B-RFP and AAV.CPP.16-CAG-H2B-RFP totransduce various tissues in mice was also evaluated. 1×10¹¹ vg wasinjected intravenously. The number of cells transduced was normalized tothe number of total cells labeled by DAPI nuclear staining. The resultsshowed that AAV.CPP.16 was more efficient than AAV9 in targeting heart(FIG. 9A); skeletal muscle (FIG. 9B), and dorsal root ganglion (FIG. 9C)tissue in mice.

Example 7. BBB Permeation in a Non-Human Primate Model

2×10¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injectedintravenously into 3-month-old cynomolgus monkeys. AAV-transduced cells(shown in black) were visualized using antibody staining against AADC.As shown in FIGS. 10A-D, AAV.CPP.16 and AAV.CPP.21 showed enhancedability vs. AAV9 to transduce brain cells after intravenousadministration in non-human primates.

AAV.CPP.16 transduced significantly more cells then wt AAV9 in theprimary visual cortex (FIG. 10A), parietal cortex (FIG. 10B), thalamus(FIG. 10C), and cerebellum (FIG. 10D). See Materials and Methods #7-8for more details.

Example 8. AAV.CPP.16 and AAV.CPP.21 do not Bind to LY6A

LY6A serves as a receptor for AAV.PHP.eB and mediates AAV.PHP.eB'srobust effect in crossing the BBB in certain mouse strains.Over-expressing mouse LY6A in cultured 293 cells significantly increasedbinding of AAV.PHP.eB to the cell surface (see FIG. 11A). On thecontrary, over-expressing LY6A does not increase viral binding for AAV9,AAV.CPP.16 or AAV.CPP.21 (see FIG. 11B). This suggests AAV.CPP.16 orAAV.CPP.21 does not share LY6A with AAV.PHP.eB as a receptor. SeeMaterials and Methods #9 for more details.

Example 9. Delivering Therapeutic Proteins to the Brain Using AAV.CPP.21

AAV.CPP.21 was used to systemically deliver the “suicide gene” HSV.TK1in a mouse model of brain tumor. HSV.TK1 turns the otherwise “dormant”ganciclovir into a tumor-killing drug. Intravenously administeredAAV.CPP.21-H2BmCherry (FIG. 12A, bottom left and middle right panel) wasshown to target tumor mass, especially the tumor expanding frontier. Asshown in FIGS. 12B-C, using AAV.CPP.21 to systemically deliver the“suicide gene” HSV.TK1 resulted in shrinkage of brain tumor mass, whencombined with the pro-drug ganciclovir. These results show thatAAV.CPP.21 can be used to systemically deliver a therapeutic gene intobrain tumor. See Materials and Methods #10 for more details.

Example 10. Intracerebral Administration of AAV.CPP.21

In addition to systemic administration (such as in Example 2), an AAV asdescribed herein was administered locally into the mouse brain.Intracerebral injection of AAV9-H2B-RFP and AAV.CPP.21-H2B-RFP (FIG. 13) resulted in more widespread and higher-intensity RFP signal inAAV.CPP.21-treated brain sections vs. AAV9-treated ones. See Materialsand Methods #4 for more details.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An adeno-associated virus (AAV) vector comprisingan AAV capsid, wherein the AAV capsid comprises a peptide insert of upto 21 amino acids, and wherein the peptide insert comprises 5-7 aminoacids of TVSALFK (SEQ ID NO: 8).
 2. The AAV vector of claim 1,comprising a transgene sequence.
 3. The AAV vector of claim 2, whereinthe transgene sequence encodes a therapeutic agent.
 4. The AAV vector ofclaim 1, comprising a non-coding RNA.
 5. The AAV vector of claim 4,wherein the non-coding RNA is shRNA, siRNA or miRNA.
 6. The AAV vectorof claim 2, wherein delivery of the transgene sequence to an organ ortissue is enhanced relative to an AAV vector comprising an AAV capsidwithout the peptide insert and the transgene sequence.
 7. The AAV vectorof claim 6, wherein the organ or tissue comprises permeability barriers.8. The AAV vector of claim 6, wherein the organ or tissue comprisesepithelium comprising tight junctions.
 9. The AAV vector of claim 6,wherein the organ or tissue is the brain or central nervous system. 10.The AAV vector of claim 2, wherein delivery of the non-coding RNA to anorgan or tissue is enhanced relative to an AAV vector comprising an AAVcapsid without the peptide insert and the non-coding RNA.
 11. The AAVvector of claim 10, wherein the organ or tissue comprises permeabilitybarriers.
 12. The AAV vector of claim 10, wherein the organ or tissuecomprises epithelium comprising tight junctions.
 13. The AAV vector ofclaim 10, wherein the organ or tissue is the brain or central nervoussystem.
 14. A composition comprising the AAV vector of claim 1, and apharmaceutically acceptable carrier.
 15. A composition comprising theAAV vector of claim 2, and a pharmaceutically acceptable carrier.
 16. Acomposition comprising the AAV vector of claim 3, and a pharmaceuticallyacceptable carrier.
 17. A composition comprising the AAV vector of claim4, and a pharmaceutically acceptable carrier.
 18. A compositioncomprising the AAV vector of claim 5, and a pharmaceutically acceptablecarrier.